Method of reducing the risk of retinopathy of prematurity in preterm infants

ABSTRACT

Disclosed is a method of reducing the risk or severity of retinopathy of prematurity in preterm infants. The method comprises (a) measuring skin carotenoid levels in preterm infants, preferably by Raman Spectroscopy, and then (b) administering supplemental carotenoids to those infants in need thereof, wherein the supplemental carotenoids comprise lutein, lycopene, beta-carotene, and zeaxanthin. The supplemental carotenoids may be provided by an infant formula comprising, on a ready-to-feed basis, from about 100 to about 2000 mcg/liter of total carotenoids, wherein the total carotenoids include at least about 50 mcg/liter of lutein. The formulas may further comprise docosahexaenoic acid.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. Patent ApplicationNo. 11/584,704, filed Oct. 20, 2006, which makes reference to and claimsthe benefit of U.S. Provisional Patent Application 60/730,283 filed Oct.26, 2005.

TECHNICAL FIELD

The present invention relates to infant formulas and methods for usingthose formulas to promote retinal health and vision development ininfants, including reducing the severity or risk of retinopathy ofprematurity.

BACKGROUND OF THE INVENTION

Infant formulas are commonly used today to provide supplemental or solesource nutrition early in life. These formulas contain protein,carbohydrate, fat, vitamins, minerals, and other nutrients. They arecommercially available as powders, ready-to-feed liquids, and liquidconcentrates.

Although many infant formulas provide a quality alternative to humanmilk, they still do not provide the same high level of nutrition asfound in human milk As such, much of the research effort into infantformulas over the past several years has been directed to betterunderstanding the natural constituents of human milk, and then modifyinginfant formulas accordingly, or at least to the extent possible withcurrently available technology.

Arachidonic acid and docosahexaenoic acid, for example, have beenidentified in human milk and subsequently added to synthetic infantformulas. These fatty acids support brain and vision development ininfants, and are now commonly found in commercially available formulassuch as Similac® Advance® Infant Formula, Isomil® Advance® Infantformula, and Similac® Special Care® Advance® infant formula, all ofwhich are available from Ross Products Division, Abbott Laboratories,Columbus, Ohio, USA.

Lutein has also been identified in human milk Although it is notcurrently added to infant formulas as an isolated ingredient, lutein canbe found at low concentrations in infant formulas as an inherentingredient in some of the natural oils commonly used make such formulas.Lutein is an antioxidant that also happens to concentrate within theretina of the eye. It is generally known that dietary lutein may provideindividuals with eye health benefits, and it is speculated that suchbenefits may be extended to infants receiving lutein from either humanmilk or supplemented infant formula

It is now believed that a combination of lutein and docosahexaenoic acidmay be particularly important in promoting retinal health and visiondevelopment in infants. Both materials are present in human milk andboth are known to concentrate in the retina in otherwise healthysubjects. Docosahexaenoic acid (DHA), as a polyunsaturated fatty acid,is highly susceptible to damage by oxidation and degradation within theeye, while lutein is a known antioxidant It is believed that by addinglutein to infant formulas, not only will it concentrate within theretina, it may also reduce oxidative degradation of the retinal DHA andthus further promote retinal health and vision development in the infant

It has now been found, however, that lutein concentrations in infantformula must be much higher than the lutein concentrations found inhuman milk in order to achieve the same plasma lutein concentrationsfound in breast fed infants due to a lower relative bioavailability oflutein from infant formula Although infant formulas today typicallycontain less than about 20 mcg/liter of lutein, most of which comesinherently from added fats and oils, it has now been found that suchlutein concentrations must exceed about 50 mcg/liter, preferably fromabout 100 mcg/liter to about 200 mcg/liter, in order to duplicate plasmalutein concentrations found in exclusively in breast fed infants.

Consequently, it has also been found that infant formulas containingcombinations of lutein and DHA, as described above, should now beformulated with higher ratios (lutein to DHA) than are commonly found inhuman milk. These weight ratios of lutein (mcg) to DHA (mg) should nowrange from about 1:2 to about 10:1.

It has also been found that the severity or risk of retinopathy ofprematurity (ROP) is influenced by skin carotenoid levels found inpreterm infants fed human milk This finding suggests that preterminfants can be monitored for skin carotenoid concentrations, and thenfor those infants demonstrating low skin carotenoid levels, supplementalcarotenoids may be administered in amounts sufficient to increase skincarotenoid levels, to thus reduce the risk or severity of retinopathy ofprematurity.

SUMMARY OF THE INVENTION

A first embodiment is a method of reducing the risk or severity ofretinopathy of prematurity in preterm infants. The method comprises (a)measuring skin carotenoid levels in preterm infants, preferably by RamanSpectroscopy, and then (b) administering supplemental carotenoids tothose infants in need thereof, wherein the supplemental carotenoidscomprise lutein, lycopene, beta arotene, and zeaxanthin. Thesupplemental carotenoids may be provided by an infant formulacomprising, on a ready-to-feed basis, from about 100 to about 2000mcg/liter of total carotenoids, wherein the total carotenoids include atleast about 50 mcg/liter of lutein.

The supplemental carotenoids may also be administered as a preterminfant formula comprising fat, protein, carbohydrate, vitamins, andminerals. The infant formula may further comprise docosahexaenoic acidin a weight ratio of lutein (mcg) to docosahexaenoic acid (mg) of fromabout 1:2 to about 10:1.

It has been found that the severity or risk of retinopathy ofprematurity (ROP) is influenced by skin carotenoid levels found inpreterm infants fed human milk. This finding suggests that skincarotenoid levels may be monitored in preterm infants, and thenadministered to those infants in need thereof to reduce the risk orseverity of retinopathy of prematurity.

It has been found that infant formulas may be prepared with luteinconcentrations of at least 50 mcg/liter if they are to produce the sameplasma lutein concentrations found in breast fed infants, even thoughhuman milk itself typically contains no more than about 30 mcg/liter oflutein. It has also been found, consequently, that the weight ratio oflutein (mcg) to DHA (mg) in an infant formula may range from about 1:2to about 10:1. It is believed that the combination of lutein anddocosahexaenoic acid are particularly useful in promoting retinal healthand vision development in infants, provided that sufficient quantitiesof each are designed into the infant formula as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of lutein intake (mcg/day) and corresponding plasmalutein concentrations (mcg/dl) in infant groups fed human milk (HM) orinfant formulas containing varied concentrations of lutein [CTRL with14.6 mcg lutein/liter (no added lutein, all lutein inherent iningredients)]; L1 with 32.6 mcg lutein/liter (approximately 18 mcg/literadded lutein, remainder inherent), L2 with 52.6 mcg lutein/liter(approximately 38 mcg/liter added lutein, remainder inherent).

FIG. 2 is a graph showing visual acuity as measured by sweep visualevoked potential (logMAR) in monkeys at 4, 8, and 12 weeks of age. Themonkeys are fed infant formula with either DHA and added lutein (n=8) orDHA without added lutein (n-8) during a 12 week feeding period.

DETAILED DESCRIPTION OF THE INVENTION

The present invention, including essential and some optional features orlimitations, is described hereinafter in detail.

The terms “infant” and “preterm infant” as used herein, unless otherwisespecified, are used interchangeably to mean preterm infants born at lessthan about 37 weeks gestation, typically from about 26 to about 34 weeksgestation, and or low birth weight infants (term or preterm) at lessthan 2.5 kg at birth, including those less than 1.8 kg at birth.

The term “infant formula” as used herein, unless otherwise specified,refers to a nutritional composition, typically free of eggphospholipids, that is designed for infants and contains sufficientprotein, carbohydrate, fat, vitamins, and minerals to potentially serveas the sole source of nutrition when provided in sufficient quantity.

The term “ready-to-feed” as used herein, unless otherwise specified,refers to infant formulas in liquid form suitable for administration toan infant, including reconstituted powders, diluted concentrates, andmanufactured liquids.

As used herein, all concentrations expressed as either “mcg/liter” or“mg/liter” refer to ingredient concentrations within the describedinfant formulas as calculated on a ready-to-feed or as fed basis, unlessotherwise specified.

All percentages, parts and ratios as used herein are by weight of thetotal composition, unless otherwise specified All such weights as theypertain to listed ingredients are based on the active level and,therefore, do not include solvents or by-products that may be includedin commercially available materials, unless otherwise specified.

All references to singular characteristics or limitations of the presentinvention shall include the corresponding plural characteristic orlimitation, and vice versa, unless otherwise specified or clearlyimplied to the contrary by the context in which the reference is made.

All combinations of method or process steps as used herein can beperformed in any order, unless otherwise specified or clearly implied tothe contrary by the context in which the referenced combination is made.

The infant formulas and methods described herein may also besubstantially free of any optional or selected essential ingredient orfeature described herein, provided that the remaining formula stillcontains all of the required ingredients or features as describedherein. In this context, the term “substantially free” means that theselected composition contains less than a functional amount of theoptional ingredient, typically less than 0.1% by weight, and alsoincluding zero percent by weight of such optional or selected essentialingredient.

The methods of the present invention may comprise, consist of, orconsist essentially of the essential elements and limitations of theinvention described herein, as well as any additional or optionalingredients, components, or limitations described herein or otherwiseuseful in nutritional formula applications.

Methods of Use

A first embodiment of the present invention is a method of reducing therisk or severity of retinopathy of prematurity in preterm infants. Themethod comprises (a) measuring skin carotenoid levels in preterminfants, preferably by Raman Spectroscopy, and then (b) administeringsupplemental carotenoids to those infants in need thereof, wherein thesupplemental carotenoids comprise lutein, lycopene, beta-otene, andzeaxanthin. The supplemental carotenoids may be provided by an infantformula comprising, on an as-fed basis, up to about 2000 mcg/liter oftotal carotenoids, wherein the total carotenoids include at least about50 mcg/liter of lutein.

The term “supplemental carotenoids” as used herein, unless otherwisespecified, means a carotenoid blend comprising up to about 2000mcg/liter, including from about 50 to about 1500 mcg/liter, and alsoincluding from about 100 to about 1200 mcg/liter, of total carotenoids,wherein the total carotenoids comprise beta-carotene, lycopene,zeaxanthin, and lutein, which includes at least about 50 mcg/liter oflutein, including from about 50 to about 1150 mcg/liter, also includingfrom about 75 to about 230 mcg/liter, and also including from about 100to about 200 mcg/liter.

This particular method may therefore provide the infant with aneffective amount of lutein to provide the stated benefits, includingfrom about 7 to about 300 mcg/kg/day, including from about 14 to about220 mcg/kg/day, and also including from about 22 to about 150 mcg/kg/day(of lutein per kg of body weight of the infant).

All lutein and carotenoid concentrations and ratios referenced hereinare calculated on a free lutein or carotenoid basis, unless otherwisespecified.

The supplemental carotenoids may also be administered alone or incombination with other ingredients as a preterm infant formulacomprising fat, protein, carbohydrate, vitamins, and minerals. Theinfant formula may further comprise docosahexaenoic acid in a weightratio of lutein (mcg) to docosahexaenoic acid (mg) of from about 1:2 toabout 10:1, including from about 1.5:1 to about 9:1, also including fromabout 1.7:1 to about 5:1.

When supplemental carotenoids are administered in the form of a preterminfant formula, that formula may be prepared and administered as a solesource, primary source, or supplemental source of nutrition.

Eye and vision development occurs at a rapid rate during the first yearof life. At birth, infants can only see high-contrast objects at perhaps25-30 cm away. During the next 6 months, the infant's retina developsenough to see and discern small details. And as an infant's visiondevelops, most of which will occur during the first year, the infantbecomes better able to learn through visual stimulation now madepossible with a newly developed sight. For infants, this visual learningthen plays a key role in brain and cognitive development, especiallyduring the first 2-3 years of life.

By promoting retinal health and vision development in infants, theinfant formulas of the present invention may also help children developtheir ability to visually learn as soon as possible, and to potentiallyaccelerate brain and cognitive development associated with early visualstimulation through the developing retina of the eye. The infantformulas described herein are therefore useful in promoting visiondevelopment in infants, and consequently are useful in promotingsecondary benefits such as associated cognitive and brain developmentthrough early visual stimulation. It is also useful in preterm infantsto help accelerate the development of normal vision, to thus reduce thetime needed to catch-up with development milestones set by their terminfant counterparts.

The methods described herein are particularly useful to reduce the riskor severity of retinopathy of prematurity. This condition often affectspreterm infants and is most commonly characterized by abnormaldevelopment of retinal vessels in the eye possibly as a result ofoxidative stress secondary to high oxygen tension. This affliction canoccur to varying degrees, from slight vessel involvement with minimal orno impact on vision, to partial or complete retinal detachment leadingto blindness. Historically, therapy for appropriate cases included lasertreatment as well as cryotherapy.

In accordance with the methods hereof, the supplemental carotenoids maybe administered to those infants with a measured Raman Count of lessthan about 20,000, including from about 100 to about 15,000, alsoincluding from about 400 to about 10,000, wherein all such ranges areproceeded by administration of supplemental carotenoids in amountssufficient to increase the Raman Count in the infant, typically by atleast about 5%, including from about 20% to about 500%. Raman countscorrelate with serum and tissue carotenoid levels.

Carotenoids

The supplement carotenoids for us herein—lutein, lycopene,beta-carotene, and zeaxanthin—may be provided or otherwise administeredin any form suitable for use in preterm infants.

The term “lutein” as used herein, unless otherwise specified, refers toone or more of free lutein, lutein esters, lutein salts, or other luteinderivatives or related structures as described or otherwise suggestedherein Lutein or lutein sources suitable for use in the infant formulasof the present invention include free lutein as well as esters, salts orother derivatives or related structures thereof, including those thatconform to the formula:

The above formula includes the general structure of lutein and relatedderivatives or structures. Free lutein, for example, corresponds to theformula wherein R₁ and R₂ are both hydrogen, and includes cis and transisomers thereof as well as salts thereof, e.g., sodium, potassium.

Lutein esters suitable for use herein include any lutein ester of theabove formula wherein R₁ and R₂ are the same or different, and arenutritionally acceptable monovalent salts, hydrogen or an acyl residueof a carboxylic acid, provided that at least one of R₁ or R₂ is an acylresidue of a carboxylic acid. Suitable lutein esters include, as well,both cis and trans isomers. The R₁ and R₂ moieties are residues of asaturated or unsaturated C₁ to C₂₂ fatty carboxylic acids, non-limitingexamples of which include formic, acetic, propionic, butyric, valeric,caproic, caprylic, capric, lauric, myristic, palmitic, stearic, andoleic acids.

Lutein for use herein includes any natural or synthetic source that isknown for or is otherwise an acceptable source for use in oralnutritionals, including infant formulas. Lutein sources can be providedas individual ingredients or in any combination with other materials orsources, including sources such as multivitamin premixes, mixedcarotenoid premixes, pure lutein sources, and inherent lutein from otherfat or oil components in the infant formula The lutein concentrationsand ratios as described herein are calculated based upon added andinherent lutein sources. The infant formulas of the present inventionpreferably comprise at least about 25%, more preferably from about 50%to about 95%, by weight of total lutein as added lutein, the remainderbeing inherent lutein that accompanies added fats and oils.

Each of the carotenoids for use herein may be obtained from any known orotherwise suitable material source for use in infant nutritionalformulas, and each can be provided individually, or all together, or inany combination and from any number of sources, including sources suchas multivitamin premixes containing other vitamins or minerals incombination with one or more of the other carotenoids as describedherein. Non-limiting examples of some suitable sources include LycoVit®synthetic lycopene dispersion in vegetable oil provided by BASF, MountOlive, N.J., USA, Lyc-O-Mato® tomato extract in oil, powder, or beadform provided by LycoRed Natural Products Industries, Ltd., Bear Sheba,Israel, water-dispersible and oil soluble lycopene provided by DSMNutritional Products, Basel, Switzerland, FloraGLO® brand free luteinprovided by Kemin Foods, Des Moines, Iowa, Xangold® Lutein Estersprovided by Cognis, Cincinnati, Ohio, U.S.A., and beta-carotene providedby BASF, Mount Olive, N.J.

Non-limiting examples of some suitable lutein sources for use hereininclude FloraGLO® Crystalline Lutein, available from Kemin Foods, DesMoines, Iowa, USA; and Xangold® Lutein Esters provided by Cognis,Cincinnati, Ohio, USA.

The infant formulas for use herein include those comprising a singlesource combination of free lutein and zeaxanthin, in a purifiedcrystalline extract from the marigold flower (Tagetes erecta), whereinthe free lutein represents from 85% to 95% by weight of the combinationand the zeaxantiin represents from about 5% to about 15% by weight ofthe combination. A lutein-zeaxanthin combination is available from KeminFoods, Des Moines, Iowa, USA, under the FloraGLO® brand.

Docosahexaenoic Acid (DHA)

The infant formulas for use herein may comprise docosahexaenoic acid, anorganic carboxylic acid having a chain length of 22 carbons with 6double bonds beginning with the third carbon from the methyl end (22:6n-3). Any source of docosahexaenoic acid is suitable for use hereinprovided that such a source is also known for or otherwise suitable foruse in infant formulas and is compatible with the other selectedingredients in the formula.

Docosahexaenoic acid concentrations in the infant formulas may beselected so that the resulting weight ratio of lutein to docosahexaenoicacid falls within the range as defined herein. Such concentrations mosttypically range from about 36 to 360 mg/liter, including from about 50to about 144 mg/liter, and also including from about 72 to about 130mg/liter, as calculated on a ready-to-feed basis.

The docosahexaenoic acid may be added to the infant formula as freefatty acids or as compounds or materials that can otherwise provide asource of such free fatty acids upon or following administration to theinfant, including non-egg phospholipids and glyceride esters (mono-,di-, tri-) of docosahexaenoic acids. Polyunsaturated fatty acids andsources thereof are described in U.S. Pat. No. 6,080,787 (Carlson, etal.) and U.S. Pat. No. 6,495,599 (Auestad, et al.), which descriptionsare incorporated by reference herein. Some non-limiting examples ofsuitable docosahexaenoic acid sources include fish oils, algal oils,other single cell oils, and combinations thereof.

The infant formulas may further comprise, in addition to thedocosahexaenoic acid as described herein, other long chainpolyunsaturated fatty acids such as arachidonic acid (20:4 n-6),eicosapentaenoic acid or EPA (20:5 n-3), linoleic acid (18:2 n-6),γ-linolenic acid or GLA (18:3 n-6), α-linolenic acid (18:3 n-3),dihomo-γ-linolenic or DHGLA (20:3 n-6), α-linolenic (18:3 n-3),stearidonic acid (18:4 n-3), and combinations thereof. Such optionallong chain polyunsaturated fatty acids may likewise be formulated intothe infant formula as free fatty acids or as compounds or materials thatcan otherwise provide a source of such free fatty acids upon orfollowing administration to the infant, including non-egg phospholipidsand glyceride esters (mono-, di-, tri-) of docosahexaenoic acids.

Other Nutrients

The infant formulas for use herein may comprise fat, protein,carbohydrate, minerals, and vitamins, all of which are selected in kindand amount to meet the dietary needs of the intended infant population.

Many different sources and types of carbohydrates, fats, proteins,minerals and vitamins are known and can be used in the infant formulas,provided that such nutrients are compatible with the added ingredientsin the selected formulation and are otherwise suitable for use in aninfant formula

Carbohydrates suitable for use herein may be simple or complex,lactose-containing or lactose-free, or combinations thereof,non-limiting examples of which include hydrolyzed, intact, naturallyand/or chemically modified cornstarch, maltodextnin, glucose polymers,sucrose, corn syrup, corn syrup solids, rice or potato derivedcarbohydrate, glucose, fructose, lactose, high fructose corn syrup andindigestible oligosaccharides such as fructooligosaccharides (FOS),galactooligosaccharides (GOS), and combinations thereof.

Proteins suitable for use herein include hydrolyzed, partiallyhydrolyzed, and non-hydrolyzed or intact proteins or protein sources,and can be derived from any known or otherwise suitable source such asmilk (e.g., casein, whey), animal (e.g., meat, fish), cereal (e.g.,rice, corn), vegetable (e.g., soy), or combinations thereof.

Proteins for use herein may also include, or be entirely or partiallyreplaced by, free amino acids known for or otherwise suitable for use ininfant formulas, non-limiting examples of which include alanine,arginine, asparagine, carnitine, aspartic acid, cystine, glutamic acid,glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, taurine, threonine, tryptophan, taurine,tyrosine, valine, and combinations thereof These amino acids are mosttypically used in their L-forms, although the corresponding D-isomersmay also be used when nutritionally equivalent Racemic or isomericmixtures may also be used.

Fats suitable for use in the infant formulas include coconut oil, soyoil, corn oil, olive oil, safflower oil, high oleic safflower oil, algaloil, MCT oil (medium chain triglycerides), sunflower oil, high oleicsunflower oil, palm and palm kernel oils, palm olein, canola oil, marineoils, cottonseed oils, and combinations thereof.

Vitamins and similar other ingredients suitable for use in the infantformulas include vitamin A, vitamin D, vitamin E, vitamin K, thiamine,nioflavin, pyridoxine, vitamin B12, niacin, folic acid, pantothenicacid, biotin, vitamin C, choline, inositol, salts and derivativesthereof, and combinations thereof.

Minerals suitable for use in the infant formulas include calcium,phosphorus, magnesium, iron, zinc, manganese, copper, chromium, iodine,sodium, potassium, chloride, and combinations thereof.

The infant formulas preferably comprise nutrients in accordance with therelevant infant formula guidelines for the targeted consumer or userpopulation, an example of which would be the Infant Formula Act, 21U.S.C. Section 350(a).

Instant formulas for use herein may comprise the carbohydrate, fat, andprotein concentrations described in the following table. TABLE 1 InfantFormula Nutrients¹ Nutrient Range gm/100 kcal gm/liter² Carbohydrate1^(st) embodiment 8-16  54-108 2^(nd) embodiment 9-13 61-88 Fat 1^(st)embodiment 3-8  20-54 2^(nd) embodiment  4-6.6 27-45 Protein 1^(st)embodiment  1-3.5  7-24 2^(nd) embodiment 1.5-3.4  10-23¹All numerical values may be modified by the term “about”²From ready-to-feed liquid, reconstituted powder, or diluted concentrate

The infant formulas for use herein may include those formulas thatcomprise per 100 kcal of formula one or more of the following: vitamin A(from about 250 to about 750 IU), vitamin D (from about 40 to about 100IU), vitamin K (greater than about 4 mcg), vitamin E (at least about 0.3IU), vitamin C (at least about 8 mg), thiamine (at least about 8 g),vitamin B 12 (at least about 0.15 g), niacin (at least about 250 g),folic acid (at least about 4 g), pantothenic acid (at least about 300g), biotin (at least about 1.5 g), choline (at least about 7 mg), andinositol (at least about 4 mg).

These infant formulas may also comprise per 100 kcal of formula one ormore of the following: calcium (at least about 50 mg), phosphorus (atleast about 25 mg), magnesium (at least about 6 mg), iron (at leastabout 0.15 mg), iodine (at least about 5 g), zinc (at least about 0.5mg), copper (at least about 60 g), manganese (at least about 5 g),sodium (from about 20 to about 60 mg), potassium (from about 80 to about200 mg), and chloride (from about 55 to about 150 mg).

Optional Ingredients

The infant formulas for use herein may further comprise other optionalingredients that may modify the physical, chemical, aesthetic orprocessing characteristics of the compositions or serve aspharmaceutical or additional nutritional components when used in thetargeted infant population Many such optional ingredients are known orare otherwise suitable for use in nutritional products and may also beused in the infant formulas for use herein, provided that such optionalmaterials are compatible with the essential materials described hereinand are otherwise suitable for use in an infant formula.

Non-limiting examples of such optional ingredients includepreservatives, additional anti-oxidants, emulsifying agents, buffers,colorants, flavors, nucleotides and nucleosides, probiotics, prebiotics,lactoferin and related derivatives, thickening agents and stabilizers,and so forth.

Product Form

The infant formulas for use herein may be prepared as any product formsuitable for use in infants, including reconstitutable powders,ready-to-feed liquids, and dilutable liquid concentrates, which productforms are all well known in the nutrition and infant formula arts.

The infant formulas for use herein may have any caloric density suitablefor the intended infant population, or provide such a density uponreconstitution of a powder embodiment or upon dilution of a liquidconcentrate embodiment Most caloric densities are at least about 18kcal/fl oz (609 kcal/liter), more typically from about 20 kcal/fl oz(675-680 kcal/liter) to about 25 kcal/fl oz (820 kcal/liter), even moretypically from about 20 kcal/fl oz (675-680 kcal/liter) to about 24kcal/fl oz (800-810 kcal/liter). Generally, the 22-30 kcal/fl oz, mosttypically from about 22-24 kcal/fl oz, formulas are more commonly usedin pre-term of low birth weight infants, and the 20-21 kcal/fl oz(675-680 to 700 kcal/liter) formulas are more often used in terminfants. Higher caloric feedings may be used with pre-term infants oflow birth weight; such feedings are typically from about 27 kcal/fl oz(90-95 kcal/liter) to about 30 kcal/fl oz (1000-1015 kcal/liter).

For powder infant formulas for use herein, such powders are typically inthe form of flowable or substantially flowable particulate compositions,or at least particulate compositions that can be easily scooped andmeasured with a spoon or similar other device, wherein the compositionscan easily be reconstituted by the intended user with a suitable aqueousfluid, typically water, to form a liquid nutritional formula forimmediate oral or enternal use. In this context, “immediate” usegenerally means within about 48 hours, most typically within about 24hours, preferably right after reconstitution. These powder embodimentsinclude spray dried, agglomerated, dry mixed or other known or otherwiseeffective particulate form. The quantity of a nutritional powderrequired to produce a volume suitable for one serving can vary.

The infant formulas for use herein may be packaged and sealed in singleor multi-use containers, and then stored under ambient conditions for upto about 36 months or longer, more typically from about 12 to about 24months. For multi-use containers, these packages can be opened and thencovered for repeated use by the ultimate user, provided that the coveredpackage is then stored under ambient conditions (e.g., avoid extremetemperatures) and the contents used within about one month or so.

Method of Manufacture

The infant formulas for use herein may be prepared by any known orotherwise effective technique suitable for making and formulating aninfant formula or similar other formula, variations of which may dependupon variables such as the selected product form, ingredientcombination, packaging and container selection, and so forth, for thedesired infant formula Such techniques and variations for any givenformula are easily determined and applied by one of ordinary skill inthe infant nutrition formulation or manufacturing arts.

The infant formulas for use herein, including the exemplified formulasdescribed hereinafter, can therefore be prepared by any of a variety ofknown or otherwise effective formulation or manufacturing methods. Thesemethods most typically involve the initial formation of an aqueousslurry containing carbohydrates, proteins, lipids, stabilizers or otherformulation aids, vitamins, minerals, or combinations thereof The slurryis emulsified, pasteurized, homogenized, and cooled. Various othersolutions, mixtures, or other materials may be added to the resultingemulsion before, during, or after further processing. This emulsion canthen be further diluted, heat-treated, and packaged to form aready-to-feed or concentrated liquid, or it can be heat-treated andsubsequently processed and packaged as a reconstitutable powder, e.g.,spray dried, dry mixed, agglomerated.

Other suitable methods for making nutritional formulas are described,for example, in U.S. Pat. No. 6,365,218 (Borschel, et al.), U.S. Pat.No. 6,589,576 (Borschel, et al.), U.S. Pat. No. 6,306,908 (Carlson, etal.), U.S. Patent Application 20030118703 A1 (Nguyen, et al.), whichdescriptions are incorporated herein by reference.

Experiment

The purpose of this experiment is to evaluate changes in visual acuityin animals fed infant formulas comprising either DHA or DHA with addedlutein. Sixteen monkeys are fed one of two defined infant formulasduring their first 12 weeks of life. One is a control formula—Similac®Advance® Infant formula, available from Abbott Laboratories, ColumbusOhio, and the other is an experimental formula including Similac®Advance® Infant Formula as a base, but with added carotenoids comprisinglutein. The formulas include the following: Experimental NutrientControl formula formula DHA 50 mg/L 60 mg/L Lutein 0 mcg/L added (18mcg/L inherent) 117 mcg/L Zeaxanthin 0 mcg/L added (4 mcg/L inherent)  36 mcg/L Zeaxanthin/ 22% 31% lutein

The base formula (Similac® Advance® Infant formula) contains water,nonfat milk, lactose, high oleic safflower oil, soy oil coconut oil,whey protein concentrate; C. cohnii oil, K. alpina oil, potassiumcitrate, calcium carbonate, ascorbic acid, mono- and diglycerides, soylecithin, carrageenan, potassium chloride, magnesium chloride, sodiumchloride, ferrous sulfate, choline chloride, choline bitartrate,taurine, m-inositol, d-alpha-tocopheryl acetate, L arnitine, zincsulfate, niacinamide, calcium pantothenate, riboflavin, vitamin Apalmitate, cupric sulfate, thiamine chloride hydrochloride, pyridoxinehydrochloride, beta-carotene, folic acid, manganese sulfate,phylloquinone, biotin, sodium selenate, vitamin D3, cyanocobalamin andnucleotides (adenosine 5′-monophosphate, cytidine 5′-monophosphate,disodium guanosine 5′-monophosphate, disodium uridine 5′-monophosphate).

The monkeys are randomized to receive either the experimental (n=8) orcontrol (n=8) formulas from birth to 12 weeks of life. The animals donot receive any milk from their mothers. Infants and mothers areseparated at birth. During the study, the monkeys are exposed to lighthaving the intensity and spectral characteristics of sunlight for 12hours per day to simulate the light-induced oxidative stress potentiallyexperienced by infants. During the study, the monkeys are evaluated forseveral parameters, including plasma lutein concentrations and sweepvisual evoked potential (VEP).

Plasma Lutein

Plasma concentrations of lutein, lycopene, and beta carotene are notsignificantly different between the monkeys fed the control andexperimental formulas at birth (0 weeks of age). Plasma luteinconcentrations are significantly higher in monkeys fed the experimentalformula than monkeys fed the control formula at 4 (p<0.001), 8(p<0.001), and 12 (p<0.001) weeks of age. Similarly, plasma lycopeneconcentrations are significantly higher in the experimental groupcompared to the control group at 4 (p<0.001), 8 (p<0.001), and 12(p<0.001) weeks. Plasma beta-carotene concentrations are significantlygreater in the experimental formula group than the control formula groupat 4 (p=0.005) and 8 (p=0.010), but not 12 (p=0.052) weeks of age.

Visual Acuity

The monkeys are assessed for changes in visual acuity at 4, 8, and 12weeks of life. Visual acuity is measured by sweep visual evokedpotential (VEP), a method well known in the art for measuring visualevoked potential in infants. Visual acuity is measured by determiningthe smallest spatial frequency high contrast grating that evokes ameasurable response from the visual cortex. The VEP from the primarycortical visual area is recorded using small silver disk EEG electrodesplaced on the scalp with water-soluble electrode paste. The infant isheld in an experimenter's lap while it gazes at a video monitordisplaying phase-reversing black and white gratings. When necessary, theinfant's attention will be drawn to the center of the screen with smalldangling toys. During each “sweep”, the spatial frequency of the gratingwill be decreased stepwise from above to below the subject's acuitythreshold during a recording period of several seconds. The amplitude ofthe second harmonic of the VEP response, which reflects the responselinked to the stimulus reversal rate, will be plotted as a function ofspatial frequency to define the subject's acuity threshold (Neuringer M,Jeffrey B G: Visual development: neural basis and new assessmentmethods. J Pediatr 2003;143:S87-S95).

VEP scores from the study are summarized in the FIG. 1 graph. Lower VEP(logMAR) scores are indicative of better visual acuity. Although VEPscores decreased (i.e., visual acuity improved) for all monkeys duringthe 12 week testing period, as expected, the VEP scores at 8 weeks weresurprisingly lower in the experimental group (added lutein+DHA formula)than in the control group (DHA without added lutein formula) (4 weeks,p=0.412, etc.)

The data suggest accelerated development in infant monkeys fed theexperimental formula at 8 weeks of life—specifically in visual acuity asmeasured by VEP values. To extrapolate the data to human infants, theeye development in monkeys at ages of 4, 8, and 12 weeks corresponds tothe eye development in human infants at 4, 8, and 12 months,respectively. The data therefore suggests that even in a human infant,the experimental formula would improve visual acuity at between about 4and about 12 months of life.

EXAMPLES

The following examples represent specific formulas for use in themethods defined herein, each of which is given solely for the purpose ofillustration and is not to be construed as limitations of the presentinvention, as many variations thereof are possible without departingfrom the spirit and scope of the invention. All exemplified amounts areweight percentages based upon the total weight of the composition,unless otherwise specified.

Examples 1.1-1.3

The following are examples of milk based, ready-to-feed, infant formulassuitable for use in the skin carotenoid methods. Example 1.1 Example 1.2Example 1.3 Amount per Quantity per Quantity per Ingredient 454 kg 454kg 454 kg Water QS QS QS Lactose 27 kg 27 kg 27 kg ARA-containing oil0.167 kg 0.167 kg 0.167 kg (40% ARA) DHA-containing oil 0.063 kg 0.095kg 0.145 kg (40% DHA) Non-fat dry milk 11.33 kg 11.33 kg 11.33 kg Higholeic safflower oil 6.5 kg 6.5 kg 6.5 kg Mono- and di-glycerides 0.162kg 0.162 kg 0.162 kg Soybean oil 5 kg 5 kg 5 kg Whey protein 2.8 kg 2.8kg 2.8 kg Calcium carbonate 0.211 kg 0.211 kg 0.211 kg Coconut oil 4.6kg 4.6 kg 4.6 kg Citric acid 0.014 kg 0.014 kg 0.014 kg Potassiumcitrate 0.245 kg 0.245 kg 0.245 kg Ascorbic acid 178 g 178 g 178 gLecithin 162 g 162 g 162 g Magnesium chloride 25 g 25 g 25 g Potassiumchloride 88 g 88 g 88 g Ferrous sulfate 26 g 26 g 26 g Carrageenan 136 g136 g 136 g Choline chloride 25 g 25 g 25 g Nucleotide and 133 g 133 g133 g choline premix ³ Riboflavin 1 g 1 g 1 g L-Carnitine 1.5 g 1.5 g1.5 g Potassium hydroxide 998 g 998 g 998 g Water soluble 65 g 65 g 65 gvitamin premix ¹ Vitamin ADEK premix ² 21 g 21 g 21 g Vitamin A 0.4 g0.4 g 0.4 g Added carotenoids ⁴ — ⁴ — ⁴ — ⁴ Total carotenoids ⁴ 186 400555 Total Lutein 90 150 200 w/zeaxanthin (mcg/L) ⁵ Total lycopene 30 90135 Total beta carotene 66 160 220 Total DHA (mg/liter) 50 75 115¹ premix contains (per 65 g) 19.8 g taurine, 14.4 g inositol, 6.7 g zincsulfate, 4.2 g niacinamide, 2.6 g calcium pantothenate, 2.3 g ferroussulfate, 0.8 g cupric sulfate, 0.6 g thiamine, 0.3 g riboflavin, 0.26 gpyridoxine, 0.1 g folic acid, 0.07 g manganese sulfate, 0.03 g biotin,0.025 g sodium selenate, 0.002 g cyanocobalamin² premix contains (per 21 g) 4.0 g alpha-tocopherol acetate, 0.8 gvitamin A palmitate, 0.05 g phylloquinone, 0.006 g vitamin D3³ premix contains (per 133 g): 23 g choline bitartrate, 15 g 5′-CMP, 11g 5′-GMP, 10 g 5′-UMP, 6 g 5′-AMP⁴ Carotenoids added individually as lycopene, beta carotene, and lutein(with zeaxanthin) to achieve lutein/zeaxanthin, lycopene, and betacarotene totals.⁵ Lutein/zeaxanthin mixture contains 1.25% zeaxanthin by weight oflutein.

The exemplified formulas may be prepared by making at least threeseparate slurries that are later blended together, heat treated,standardized, packaged and sterilized. Initially, a carbohydrate-mineralslurry is prepared by dissolving lactose in water at 65-71° C., followedby the addition of magnesium chloride, potassium citrate, potassiumchloride, choline chloride, and citric acid. The resulting slurry isheld with agitation at 55-65° C. for not longer than eight hours untilit is later blended with the other prepared slurries.

A protein-fat slurry is prepared by combining high oleic safflower oil,soybean oil, and coconut oil at 55-60° C., followed by the addition ofvitamin ADEK premix, mono- and diglycerides, lecithin, carrageenan,vitamin A, carotenoids, ARA oil, and DHA oil. Whey protein and calciumcarbonate are then added The resulting protein-oil slurry is held undermoderate agitation at 40-43° C. for no longer than two hours until it islater blended with the other formed slurries.

The carbohydrate-mineral slurry is then combined with water and non-fatdry milk and agitated for 10 minutes. The protein-oil slurry is thenadded and the resulting mixture agitated for at least 10 minutes. Luteinand beta-carotene are then added to the blend and agitated for at least15 minutes. The pH of the resulting blend is adjusted to 6.68-6.75 withIN potassium hydroxide.

After waiting for a period of not less than one minute nor greater thantwo hours, the resulting blend is heated to 71-82° C. and dearated undervacuum, emulsified through a single stage homogenizer at 900-1100 psig,and then heated to 99-110° C., and then heated again to 146° C. forabout 5 seconds. The heated blend is passed through a flash cooler toreduce the temperature to 99-110° C. and then through a plate cooler tofurther reduce the temperature to 71-76° C. The cooled blend is thenhomogenized at 3900-4100/400-600 psig, and then held at 74-80° C. for 16seconds, and then cooled to 1-7° C. are taken for microbiological andanalytical testing. The mixture is held under agitation. 10081] Awater-soluble vitamin (WSV) solution and an ascorbic acid solution areprepared separately and added to the processed blended slurry. Thevitamin solution is prepared by adding the following ingredients to 9.4kg of water with agitation: potassium citrate, ferrous sulfate, WSVpremix, L-carnitine, riboflavin, and the nucleotide-choline premix. Theascorbic acid solution is prepared by adding potassium citrate andascorbic acid to a sufficient amount of water to dissolve theingredients. The vitamin and ascorbic acid solutions are then added tothe blend, and the pH of the blend adjusted to 7-10 with 45% potassiumhydroxide solution.

Based on the analytical results of the quality control tests, anappropriate amount of water is added to the batch with agitation toachieve the desired total solids. The product pH may be adjusted toachieve optimal product stability. The completed product is then placedin suitable containers and subjected to terminal sterilization.

The resulting formulas are fed to infants as a sole source of nutritionduring the first 6 to 12 months of life to provide each infant with7-300 μg/kg/day of lutein. The formulas provide improved retinal healthand vision development as described herein.

Examples 2.1-2.3

The following are examples of soy-based, powder, infant formulassuitable for use in the methods of the present invention. Example 2.1Example 2.2 Example 2.3 Amount per Amount per Amount per INGREDIENT 454kg 454 kg 454 kg Water QS QS QS High oleic 52.1 kg 52.1 kg 52.1 kgsafflower oil Coconut oil 35.2 kg 35.2 kg 35.2 kg Soy oil 38.1 kg 38.1kg 38.1 kg ARA-containing oil 1.3 kg 1.3 kg 1.3 kg (40% ARA)DHA-containing oil 0.381 kg 0.762 kg 0.876 kg (40% DHA) Oil soluble0.173 kg 0.173 kg 0.173 kg vitamin premix Ascorbyl palmitate 0.162 kg0.162 kg 0.162 kg Soy protein isolate 66.1 kg 66.1 kg 66.1 kg Corn syrup236.0 kg 236.0 kg 236.0 kg Calcium phosphate 8.0 kg 8.0 kg 8.0 kg (diand tribasic) Ferrous sulfate 0.138 kg 0.138 kg 0.138 kg Water soluble0.65 kg 0.65 kg 0.65 kg vitamin premix trace minerals/taurine Cholinechloride 0.23 kg 0.23 kg 0.23 kg Potassium iodide 0.0005 kg 0.0005 kg0.0005 kg Methionine 0.722 kg 0.722 kg 0.722 kg Ascorbic acid 0.72 kg0.72 kg 0.72 kg Potassium hydroxide 1.2 kg 1.2 kg 1.2 kg (45% solution)Potassium chloride 0.87 kg 0.87 kg 0.87 kg Magnesium Chloride 0.4 kg 0.4kg 0.4 kg Carnitine 0.05 kg 0.05 kg 0.05 kg Added carotenoids ¹ — ¹ — ¹— ¹ Total carotenoids 186 400 555 (mcg/liter) Total Lutein with 90 150200 zeaxanthin (mcg/liter) Total lycopene 30 90 135 (mcg/liter) Totalbeta carotene 66 160 220 (mcg/liter)¹ Individual carotenoids added as lycopene, beta-carotene, and lutein(with zeaxanthin) to achieve lutein/zeaxanthin, lycopene, andbet-carotene totals.² Lutein/zeaxanthin mixture contains 1.25% zeaxanthin by weight oflutein.

The first step in the preparation of the exemplified powder is thepreparation of the oil blend. Soy oil, coconut oil and high oleicsafflower oil are combined in a suitable container or tank at 60-65° C.with agitation. Ascorbyl palmitate and mixed tocopherols are added tothe tank, followed by the oil soluble vitamin premix, all withagitation. Beta-carotene (BASF, Mount Olive, N.J.), and lutein (Kemin,Des Moines, Iowa) and other carotenoids are added to the oil blend andagitated until well dispersed. Soy protein isolate and methionine arethen added to the oil blend, and the resulting mixture agitated and heldat 54.0-60° C. until used later during the manufacturing process.

The carbohydrate-mineral slurry is then prepared. Potassium chloride,sodium chloride, magnesium chloride, and potassium iodide are added towater 60-65° C., followed by di- and tri-calcium phosphates, all withagitation. Corn syrup is then added with agitation, and the slurry heldat 54-60° C. until used later during the manufacturing process.

The carbohydrate-mineral slurry is added to the oil blend. Additionalwater is added as necessary. The ARA and DHA oils are added to theblend. The pH of the resulting mixture is adjusted to 6.75-6.85 usingKOH solution. The adjusted mixture is then held at 54-60C underagitation for at least 15 minutes.

The resulting mixture is then heated to 74-79C and dearated undervacuum, emulsified through a single stage homogenizer at 0-2.76 Mpa,passed through a two-stage homogenizer at 6.2-7.6 MPa and 2.1-3.4 MPaThe homogenized mixture is held at 73-79C for 16 seconds and then cooledto 1-7C. Samples are taken for microbiological and analytical testingThe mixture is held under agitation.

A calcium carbonate solution may be prepared for use in adjusting thecalcium level of the mixture if outside of specification.

A vitamin stock solution containing a water soluble vitamin premix withtrace minerals and taurine is prepared. Potassium citrate and ferroussulfate are added to water at 37-66° C. The vitamin premix is then addedand the mixture agitated. The choline chloride and carnitine are addedand then the required amount of this vitamin mixture is added to thebatch.

An ascorbic acid solution is prepared and added slowly to the batch withagitation for at least 10 minutes. The batch is then preheated to 74-79°C. The batch is then held for 5 seconds at 107-111° C. using directsteam injection. The batch is then cooled to 71-82° C. before beingpumped to a spray dryer and dried to a flowable powder. The batch isthen packaged in suitable containers and sealed under a headspace ofless than 2.0% oxygen.

The exemplified powders are reconstituted with water to a caloricdensity of 676 kcal/liter. The resulting liquid formulas are fed topreterm infants as a sole source of nutrition at 7-300 μg/kg/day oflutein. The formula provides improved retinal health and visiondevelopment as described herein, and reduces the risk or severity ofretinopathy of prematurity in the infants.

Examples 3.1-3.3

The following are examples of milk-based, powder, infant formulassuitable for use in the methods described herein. Example 3.1 Example3.2 Example 33 Amount per Amount per Amount per Ingredient Name 454 kg454 kg 454 kg Water QS QS QS Soy oil 35.8 kg 35.8 kg 35.8 kg Coconut oil23.8 kg 23.8 kg 23.8 kg MCT oil (medium 32.1 kg 32.1 kg 32.1 kg chaintriglyceride) High Oleic 34.6 kg 34.6 kg 34.6 kg Safflower oil Ascorbylpalmitate 0.157 kg 0.157 kg 0.157 kg Vitamin A palmitate 0.002 kg 0.002kg 0.002 kg Vitamin ADEK 0.192 kg 0.192 kg 0.192 kg premix ¹ Mixedtocopherols 0.075 kg 0.075 kg 0.075 kg Whey protein 32.7 kg 32.7 kg 32.7kg concentrate Calcium carbonate 1.2 kg 1.2 kg 1.2 kg Lactose 54.5 kg54.5 kg 54.5 kg Corn syrup solids 117.1 kg 117.1 kg 117.1 kg Magnesiumchloride 0.724 kg 0.724 kg 0.724 kg Potassium citrate 2.8 kg 2.8 kg 2.8kg Sodium chloride 0.39 kg 0.39 kg 0.39 kg Sodium citrate 0.001 kg 0.001kg 0.001 kg Non-fat dried milk 116.9 kg 116.9 kg 116.9 kg Calciumphosphate 1.8 kg 1.8 kg 1.8 kg tribasic ARA-containing oil 1.3 kg 1.3 kg1.3 kg (40% ARA) DHA-containing oil 0.43 kg 0.65 kg 1.00 kg (40% DHA)Ascorbic acid 1.29 kg 1.29 kg 1.29 kg Potassium hydroxide 9.8 kg 9.8 kg9.8 kg 1N solution Ferrous sulfate 0.168 kg 0.168 kg 0.168 kg Carnitine0.136 kg 0.136 kg 0.136 kg Choline chloride 0.182 kg 0.182 kg 0.182 kgVit. and trace 0.825 kg 0.825 kg 0.825 kg mineral premix ² Inositol0.734 kg 0.734 kg 0.734 kg Nucleotide, choline 1.1 kg 1.1 kg 1.1 kgbitartrate premix ³ Added carotenoids ⁴ — ⁴ — ⁴ — ⁴ Total carotenoids186 400 555 (mcg/liter) Total Lutein with 90 150 200 zeaxanthin(mcg/liter) Total lycopene 30 90 135 (mcg/liter) Total beta carotene 66160 220 (mcg/liter)¹ premix provides 71 gm d-alpha-tocopheryl acetate, 7.29 gm Vitamin Apalmitate, 0.422 gm phylloquinone, and 0.051 gm Vitamin D3 to theproduct.² premix provides 252 gm taurine, 183 gm inositol, 84.5 gm zinc sulfate,53.8 gm niacinamide, 32.6 gm calcium pantothenate, 29 gm ferroussulfate, 10.1 gm cupric sulfate, 8.4 gm thiamine, 3.7 gm riboflavin, 3.4gm pyridoxine (HCl), 1.1 gm folic acid, 1.0 gm manganese sulfate, 0.3 gmbiotin, 0.2 gm sodium selenate, and 0.03 gm cyanocobalamin to theproduct.³ premix provides 188 gm choline bitartrate, 118 gm cytidine5′-monophosphate, 92 gm disodium guanosine 5′-monophosphate, 80 gmdisodium uridine 5′-monophosphate, and 51 gm adenosine 5′-monophosphateto the product.⁴ Individual carotenoids added as lycopene, beta-carotene, and lutein(with zeaxanthin) to achieve lutein/zeaxanthin, lycopene, andbet-carotene totals.⁵ Lutein/zeaxanthin mixture contains 1.25% zeaxanthin by weight oflutein.

This powder formula is manufactured by preparing at least two slurriesthat are later blended together, heat treated, standardized, spray driedand packaged. Initially, a carbohydrate-mineral slurry is prepared(45-50% solids) by dissolving lactose in water at 66-76° C. Corn syrupsolids are then added and allowed to dissolve, followed by the additionof magnesium chloride, potassium citrate, sodium chloride, cholinechloride, and sodium citrate, all with agitation. The resultingcarbohydrate-mineral slurry is held at 54-60° C. under agitation untilused later during the manufacturing process.

A protein-fat slurry is prepared by combining high oleic safflower oil,coconut oil, soy oil, and MCT oil at 40.5-49° C., followed by ascorbylpalmitate, mixed tocopherols, vitamin A palpitate, and the vitamin ADEKpremix, all with agitation. Lutein (Kemin, Des Moines, Iowa) and othercarotenoids, are then added with agitation. Whey protein concentrate isthen added to the slurry, followed by calcium carbonate and calciumphosphate tribasic, all with agitation. The completed protein-fat slurryis held under moderate agitation at 54-60° C. for no longer than twelvehours until it is blended with the other prepared slurries.

The carbohydrate-mineral slurry is transferred to a tank in which asufficient amount of water is added to create a final blend slurry ofapproximately 50% solids. Non-fat dry milk is then added to the blendand allowed to solubilize. The protein-fat slurry is then added and theentire blend slurry is allowed to agitate for at least 15 minutes. Theresulting blend is maintained at 60-65° C. The blend pH is adjusted to6.7-6.9 with IN KOH.

After waiting for a period of not less than one minute nor greater thantwo hours, the resulting blend is heated to 71-79° C, emulsified at2.754.1 Mpa, and then heated to 115-127° C. for about 5 using directsteam injection. The heated emulsion is then flash cooled to 87-99° C.,and homogenized at 9.7-11.0/2.75-4.1 MPa. The homogenized slurry is thencooled to 1.6-7.2° C. Samples are taken for microbiological andanalytical testing. The mixture is held under agitation.

A vitamin-trace mineral solution is prepared by adding the followingingredients to the required amount of water, under agitation: potassiumcitrate, ferrous sulfate, carnitine, vitamin and the trace mineralpremix, inositol, and nucleotide and choline bitartrate premix. Thevitamin-trace mineral solution is then added to the homogenized slurryunder agitation.

An ascorbic acid solution is prepared by adding potassium citrate andascorbic acid to water with agitation, and then adding the aqueousmixture to the homogenized slurry under agitation.

The product is preheated to 65.5-77° C. The product is then held at82-90.5° C. for 5 seconds before being flash cooled to 71-82° C. andpumped to the spray dryer. The product is spray dried to produce adesired free-flowing powder. The resulting powder is packaged undernitrogen to maximize product stability and flavor.

The exemplified powders are reconstituted with water to a caloricdensity of 676 kcal/liter. The resulting liquid formulas are fed topreterm infants as a sole source of nutrition during the first 6 to 12months of life to provide 7-300 μg/kg/day of lutein. The formulaprovides improved retinal health and vision development as describedherein, and reduces the risk or severity of retinopathy of prematurityin the infants.

Examples 4.1-4.3

The following are examples of concentrated human milk fortifier liquidsfor use in the methods described herein. Example 4.1 Example 4.2 Example4.3 Ingredient Name Gm per kg Gm per kg Gm per kg Sucrose 125.5 125.5125.5 Milk protein isolate 64.6 64.6 64.6 Coconut oil 30.2 30.2 30.2Whey protein 24.4 24.4 24.4 concentrate MCT Oil 21.9 21.9 21.9 Soy Oil21.9 21.9 21.9 Tricalcium phosphate 14.4 14.4 14.4 Potassium chloride5.18 5.18 5.18 Calcium carbonate 3.44 3.44 3.44 Magnesium phosphate 3.053.05 3.05 Potassium citrate 1.32 1.32 1.32 DHA (docosahexaenoic 0.2 0.20.2 acid) oil Soy lecithin 0.756 0.756 0.756 ARA (arachidonic 0.7290.729 0.729 acid) oil Dipotassium phosphate 0.596 0.596 0.596Monopotassium 0.466 0.466 0.466 phosphate Vitamin E 0.357 0.357 0.357Sodium chloride 0.170 0.170 0.170 KOH 5% solution Q.S. Q.S. Q.S.m-Inositol 0.0698 0.0698 0.0698 Ascorbic acid 0.913 0.913 0.913 Taurine0.0663 0.0663 0.0663 Niacinamide 0.0582 0.0582 0.0582 Vitamin A 0.04940.0494 0.0494 Zinc sulfate 0.0461 0.0461 0.0461 Calcium pantothenate0.0286 0.0286 0.0286 Ferrous sulfate 0.0136 0.0136 0.0136 Cupric sulfate0.00836 0.00836 0.00836 Riboflavin 0.00763 0.00763 0.00763 Thiaminechloride HCL 0.00507 0.00507 0.00507 Pyridoxine HCL 0.00459 0.004590.00459 Folk acid 0.000778 0.000778 0.000778 Manganese Sulfate 0.0005730.000573 0.000573 Biotin 0.000507 0.000507 0.000507 Vitamin K 0.0008350.000835 0.000835 Vitamin D3 0.000235 0.000235 0.000235 Sodium selenate0.0000491 0.0000491 0.0000491 Potassium iodide 0.0000105 0.00001050.0000105 Cyanocobalamin 0.0000103 0.0000103 0.0000103 Added carotenoids¹ — ¹ — ¹ — ¹ Total carotenoids 186 400 555 (mcg/liter) Total Luteinwith 90 150 200 zeaxanthin ² (mcg/liter) Total lycopene 30 90 135(mcg/liter) Total beta carotene 66 160 220 (mcg/liter)¹ Individual carotenoids added as lycopene, beta-carotene, and lutein(with zeaxanthin) to achieve the lutein/zeaxanthin, lycopene, andbeta-carotene totals.² Lutein/zeaxanthin mixture contains 1.25% zeaxanthin by weight oflutein.

The ingredients listed in the preceding table are combined and processedto form a concentrated human milk fortifier embodiment of the presentinvention. One method of preparing such an embodiment is describedbelow.

An initial intermediate blend is prepared by heating to 32-37° C. thespecified amounts of coconut oil, MCT oil, soy oil, DHA oil and AA oil,all with agitation. A soy lecithin emulsifier is added with agitation tothe heated blend and allowed to dissolve. Vitamins A, D, and K, NaturalVitamin E, and lutein are then added with agitation to the developingblend. Milk protein isolate (25.8 kg) and the specified amounts of ultramicronized tricalcium phosphate and calcium carbonate are added to theblend. The resulting intermediate blend is maintained at 26-48° C. undermoderate agitation for a period of time not to exceed six hours beforebeing added to the aqueous protein blend described below.

An aqueous protein blend is then prepared by heating 573 kg ofingredient water at 48-60° C., and then adding to it with agitation milkprotein isolate (38.8 kg) and the specified amount of whey proteinconcentrate. Thereafter, and with agitation, the entire intermediateblend described above is added to the aqueous protein blend. Thefollowing ingredients are then added to the resulting blend in thefollowing order: potassium citrate, dipotassium phosphate, monopotassiumphosphate, magnesium phosphate, sodium chloride, potassium chloride,potassium iodide and sucrose. After no less than five minutes, the blendpH is adjusted to 6.60-6.80 using a 1N KOH solution, and thereaftermaintained at 51-60° C., for a period of time not to exceed two hoursbefore further processing.

The pH adjusted blend is then homogenized using one or more in-linehomogenizers at pressures from 1000-4000 psig with or without a secondstage homogenization from 100-500 psig followed by heat treatment usinga HTST (high temperature short time, 74° C. for 16 seconds) or UHTST(ultra-high temperature short time, 132-154° C. for 5-15 seconds)process. The choice of UHTST or HTST is normally made based upon areview of the bioburden of each of the ingredients in the formulation.After the appropriate heat treatment, the batch is cooled in a platecooler to 1.0-5.0° C. and then transferred to a refrigerated holdingtank, where it is subjected to analytical testing and then standardizedto finished product specifications, which includes the addition of anascorbic acid solution and a water-soluble vitamin and trace mineralsolution, all of which is prepared separately before adding to thepreviously described refrigerated batch

The ascorbic acid solution is prepared by adding the specified amount ofascorbic acid to 11.1 kg of 1N KOH solution with agitation. Thewater-soluble vitamin and trace mineral solution is prepared by heating25.2 kg of ingredient water to 37° C. to 48° C. The water solublevitamins and trace minerals are added to the water as a premix whichcontains m-inositol, taurine, niacinamide, zinc sulfate, calciumpantothenate, ferrous sulfate, cupric sulfate, riboflavin, thiaminehydrochloride, pyridoxine hydrochloride, folic acid, manganese sulfate,biotin, sodium selenate, and cyanocobalamin. As noted above, bothsolutions are then added to the refrigerated batch, all with agitation.As part of batch standardization, the appropriate amount of ingredientdilution water is then added to the batch for a target total solidslevel of 31% and the pH adjusted to 7.1 with a 1N KOH solution. Thebatch is filled into suitably sized containers containing 5 ml ofproduct.

The exemplified human milk fortifier concentrates are combined withhuman milk (5 ml concentrate with 20-25 ml human milk). The fortifiedhuman milk is then fed to pre-term infants to provide 7-300 μg/kg/day oflutein. The formula provides improved retinal health and visiondevelopment as described herein, including reduced risk of retinopathyof prematurity.

Examples 5.1-5.3

This example illustrates a ready-to-feed, preterm infant formulaembodiment of the present invention. This formula is similar to Similac®Special Care® Advance® with Iron Premature Infant formula, a preterminfant formula available from Abbott Laboratories, Columbus, Ohio,except for the increased lutein concentrations and subsequent lutein todocosahexaenoic acid ratios.

The preterm infant formula includes nonfat milk, corn syrup solids,lactose, medium chain triglycerides, whey protein concentrate, soy oil,coconut oil, C. cohnii oil (source of docosahexaenoic acid), M. alpinaoil (source of arachidonic acid), calcium phosphate, calcium carbonate,potassium citrate, ascorbic acid, magnesium chloride, soy lecithin,mono- and diglycerides, m-inositol, sodium citrate, carrageenan, ferroussulfate, choline bitartrate, taurine, choline chloride, niacinamide,d-alpha-tocopheryl acetate, L-carnitine, zinc sulfate, potassiumchloride, potassium phosphate dibasic, calcium pantothenate, cupricsulfate, vitamin A palmitate, riboflavin, thiamine chloridehydrochloride, pyridoxine hydrochloride, folic acid, beta-carotene,manganese sulfate, biotin, phylloquinone, sodium selenate, vitamin D3,cyanocobalamin and nucleotides (cytidine 5′-monophosphate, disodiumguanosine 5′-sphate, monophosphate, disodium uridine 5′-monophosphate,adenosine 5′-monophosphate). A carotenoid supplement is added to theformula.

The above-referenced ingredients are formulated together by conventionalmethods to provide the following nutrition profile: Example 5.1 Example5.2 Example 5.3 Amount per Amount per Amount per 100 kcal 100 kcal 100kcal Nutrients (or 123 ml) (or 123 ml) (or 123 ml) Protein (from nonfat3.00 g 3.00 g 3.00 g milk, whey protein concentrate) Fat (form50:30:18.3 5.43 g 5.43 g 5.43 g mix of MCT oil, soy oil, and coconutoils; —mg DHA, —mg ARA; 700 mg linoleic acid) Carbohydrate 10.3 g 10.3 g10.3 g (source - 50:50 mix of corn syrup solids, lactose) Lutein 18.4mcg 37 mcg 9141 mcg Vitamin A 1250 IU 1250 IU 1250 IU Vitamin D 150 IU150 IU 150 IU Vitamin E 4.0 IU 4.0 IU 4.0 IU Vitamin K 12 mcg 12 mcg 12mcg Thiamine 250 mcg 250 mcg 250 mcg Riboflavin 620 mcg 620 mcg 620 mcgVitamin B6 250 mcg 250 mcg 250 mcg Vitamin B12 0.55 mcg 0.55 mcg 0.55mcg Niacin 5000 mcg 5000 mcg 5000 mcg Folic acid 37 mcg 37 mcg 37 mcgPantothenic acid 1900 mcg 1900 mcg 1900 mcg Biotin 37.0 mcg 37.0 mcg37.0 mcg Ascorbic acid 37 mg 37 mg 37 mg Choline 10 mg 10 mg 10 mgInositol 40.0 mg 40.0 mg 40.0 mg Calcium 180 mg (9.0 180 mg (9.0 180 mg(9.0 mEq) mEq) mEq) Phosphorus 100 mg 100 mg 100 mg Magnesium 12.0 mg12.0 mg 12.0 mg Iron 1.8 mg 1.8 mg 1.8 mg Zinc 1.50 mcg 1.50 mcg 1.50mcg Manganese 12 mcg 12 mcg 12 mcg Copper 250 mcg 250 mcg 250 mcg Iodine6 mcg 6 mcg 6 mcg Selenium 1.8 mcg 1.8 mcg 1.8 mcg Sodium 43 mg (1.9 43mg (1.9 43 mg (1.9 mEq) mEq) mEq) Potassium 129 mg (3.3 129 mg (3.3 129mg (3.3 mEq) mEq) mEq) Chloride 81 mg (2.3 81 mg (2.3 81 mg (2.3 mEq)mEq) mEq) Added — ¹ — ¹ — ¹ carotenoids ¹ Total carotenoids 186  400 555(mcg/liter) Total Lutein 90 150 200 w/zeaxanthin ² (mcg/liter) Totallycopene 30  90 135 (mcg/liter) Total beta carotene 66 160 220(mcg/liter)¹ Individual carotenoids added as lycopene, beta-carotene, and lutein(with zeaxanthin) to achieve the lutein/zeaxanthin, lycopene, andbeta-carotene totals.² Lutein/zeaxanthin mixture contains 1.25% zeaxanthin by weight oflutein.

Example 5.4

Skin carotenoid levels are measured in a preterm infant born at 26 weeksgestation, weighing 1.7 kg, age 1 day. Skin carotenoid levels aremeasured (800 Raman Counts) using non invasive Raman Spectroscopy on theinfant's palm. The infant is administered the preterm infant formula ofExample 5.2 as a primary form of daily nutrition until discharge. Theformula contains lutein, lycopene, beta carotene, and zeaxanthin. Skincarotenoids are measured periodically until discharge to assure adequatecarotenoid supplementation as noted by increasing skin carotenoid levels(increased Raman Counts). The infant subsequently experiences minimal orno signs of retinopathy of prematurity.

The exemplified ready-to-feed formulas (caloric density of 812kcal/liter) are administered to preterm infants to provide from 7-300mcg/kg of lutein per day. The administered formula improves eye healthas described herein, and are especially useful as applied to preterminfants to reduce the risk of retinopathy of prematurity and helpsprotect the eyes from natural or artificial light, especially biliarylights.

Experiment

A study is conducted to compare plasma lutein concentrations inbreastfed infants with plasma lutein concentrations in formula fedinfants. The latter received one of three formulas defined by luteinconcentrations of 32.6 mcg/liter (L1), 52.6 mcg/liter (L2), or 14.6mcg/liter (CTRL). The study groups and resulting plasma luteinconcentrations are summarized in the following table. Plasma Lutein(mcg/dL)^(‡) Feeding Group Study Day 1 Study Day 56 Control (CTRL) - noadded 1.37 ± 0.29 ^(a) 2.17 ± 0.12 ^(a) lutein Total inherent(0.40-5.10) (1.16-3.25) lutein 14.6 mcg/liter lutein) 20 18 Formula L10.78 ± 0.09 ^(a) 2.21 ± 0.16 ^(a) Added lutein approximately (0.27-2.09)(0.20-3.61) 18 mcg/liter 24 22 Inherent lutein approximately 14.6mcg/liter Total lutein 32.6 mcg/L Formula L2 0.97 ± 0.13 ^(a) 3.25 ±0.26 ^(b) Added lutein approximately (0.20-2.31) (0.60-4.70) 38mcg/liter 21 19 Inherent lutein approximately 14.6 mcg/liter Totallutein 52.6 mcg/L Human Milk (HM) 6.53 ± 0.54 ^(b) 5.88 ± 0.77 ^(c)6.5-107.8 mcg/liter (1.69-14.12)  (0.49-20.09) lutein 24 26^(‡)Values are presented as mean ± SEM, (range) n Values in a columnwith superscripts without a common letter differ(p < 0.05). See text foractual p values. Statistical comparisons among the formula groups aredone using the Kruskal-Wallis test. Comparisons between the formulagroups and the human milk group are done using the Wilcoxon rank-sumtest.

Plasma lutein concentrations (at day 56 of the study) are used as theprimary variable in the study. The primary comparison is the differencein plasma lutein concentrations between the L2 and CTRL formula groups.Secondary comparisons included differences in plasma luteinconcentrations among the formula groups (CTRL, L1, L2) and differencesbetween the formula groups and the human milk group. Plasma luteinconcentrations on day 56 from a total of 85 infants (CURL, n-18; L1,n=22; L2, n=19; HM, n=26) is used in these analyses and results reportedas mean±SEM in the following table.

Infants in the L2 formula group have significantly higher (p<0.05)plasma concentrations of lutein than infants in the L1 and CTRL formulagroups. Plasma lutein are not different between the L1 and CTRL formulagroups. The human milk group has higher plasma lutein concentrationsthan the CTRL (p<0.0001), L1 (p<0.0001), and L2 (0.0052) formula groups.Plasma lutein concentration is significantly correlated with luteinintake (r=0.436, p=0.0014). Lutein intake and plasma response from thestudy are also summarized in FIG. 1.

The data from the study show that lutein is surprisingly lessbioavailable from infant formula than from human milk (see FIG. 1). Assuch, in order for an infant formula to produce plasma luteinconcentrations in infants similar to that produced by feeding humanmilk, an infant formula must be formulated to contain at least about 50mcg/liter of lutein, preferably from 100 mcg/liter to about 200mcg/liter.

Study: Skin Carotenoids

A purpose of this study is to evaluate the relation of skin carotenoidsin the development or severity of Retinopathy of Prematurity (ROP) inhuman milk fed preterm infants. Skin carotenoid levels are measuredusing non-invasive Raman spectroscopy. It is known that skin carotenoidlevels as measured by Raman spectroscopy correlate with serum and tissuecarotenoid levels (Data et al., J Invest Dermatology, 115:441, 200; Hataet al., J Invest Dermatology 115;441, 2000; Ermakov et al., OpticsLetters, 26:1179, 2001)

Skin carotenoids are assessed non-invasively using a BioPhotonic Scanner(available from Phalmanex, Provo, Utah USA). For each infant tested, asmall area of the palm is exposed to a low-energy monochromatic laserlight of 473 nm (blue light) penetrating into the stratum comeum layerof the skin for approximately 3 minutes to obtain an adequatemeasurement Carotenoids present in the exposed area will shift thewavelength to 511 nm (green light), which is detected into a relativesignal and converted to Raman Counts (RC).

Carotenoid molecules are strong Raman scatterers when excited withwavelengths of light between 450 and 500 nanometers (nm). This range ofvisible light can be accomplished with commercially available laserlight sources. The carotenoid molecules are especially suited for thisanalysis since their characteristic double diene bonds emit strong andmeasurable Raman spectroscopic signals (511 nm) when excited with laserlight of 473 nm.

In the present study, twenty-two infants whose individual birth weights(BW) are less than 1800 g, gestation at less than 33 weeks, and fedtheir own mother's milk are enrolled at the start of feedings. Skincarotenoids are measured initially and then biweekly. Days on oxygen arerecorded Initial eye exams for ROP are initiated between 4 and 7 weeksof age. The mean gestation (±SD) is 28±2.6 weeks with mean BW of1102±356 g. Forty-one percent of the infants (9/22) develop ROP, with 3requiring laser surgery for stage 3 ROP. Infants with ROP have lowermean gestation (26 v 30 wks), BW (871 v 1262 g) and longer on oxygen(112 v 40 days) than infants with no ROP.

After controlling for prematurity, it is also discovered that infantswho develop ROP have lower initial skin carotenoid levels than infantswho had no ROP, 7.8±7.8 v 11.1±4.2 (Mann Whitney, P<0.02 ROP isnegatively associated with initial skin carotenoids(R=−0.75, P<0.001).Skin carotenoids are positively associated with birth weight (R=0.66,P<0.001), gestation (=0.69, P<0.001), day of life (R=0.64, P<0.001), butnegatively associated with days on oxygen (R=−0.57, P<0.01).

This study shows that ROP is influenced by skin carotenoid (lutein,beta-carotene, zeaxantin, lycopene) levels found in preterm infants fedhuman milk. The study suggests that preterm infants at risk ofdeveloping ROP can be monitored for skin carotenoid concentrations andmay benefit from carotenoid supplementation. This may result in reducingthe risk or severity of ROP.

1. A method of reducing the risk or severity of retinopathy ofprematurity in preterm infants, said method comprising: (a) measuringskin carotenoid levels in preterm infants, and then (b) administeringsupplemental carotenoids to those infants in need thereof, wherein thesupplemental carotenoids comprise lutein, lycopene, beta carotene, andzeaxanthin.
 2. The method of claim 1 wherein skin carotenoids levels aremeasured by Raman Spectroscopy.
 3. The method of claim 2 wherein thepreterm infants in need of supplemental carotenoids are those having aRaman Count of less than about 20,000.
 4. The method of claim 2 whereinthe supplemental carotenoids are administered in an amount effective toincrease skin carotenoids levels as measured by Raman Spectroscopy by atleast about 5%.
 5. The method of claim 1 wherein the preterm infant isborn at between about 26 and about 34 weeks gestation.
 6. The method ofclaim 1 wherein the preterm infant is administered supplementalcarotenoids in an amount effective to provide a total lutein intake,from all dietary sources, of from about 7 to about 300 mcg/kg/day. 7.The method of claim 1 wherein the preterm infant is administeredsupplemental carotenoids in an amount effective to provide a totallutein intake, from all dietary sources, of from about 14 to about 220mcg/kg/day.
 8. The method of claim 1 wherein the supplemental carotenoidis provided by an infant formula comprising, on a ready-to-feed basis,from about 100 to about 2000 mcg/liter of total carotenoids, wherein thetotal carotenoids include at least about 50 mcg/liter of lutein.
 9. Themethod of claim 8 wherein the total carotenoids include from about 100to about 1150 mcg/liter of added lutein.
 10. The method of claim 8wherein the infant formula further comprises fat, protein, carbohydrate,vitamins, and minerals, including docosahexaenoic acid in a weight ratioof lutein (mcg) to docosahexaenoic acid (mg) of from about 1:2 to about10:1.
 11. The method of claim 9 wherein the total carotenoids includefrom about 100 to about 200 mcg/liter of lutein.
 12. The method of claim11 wherein the weight ratio of lutein (mcg) to docosahexaenoic acid (mg)is from about 2:1 to about 5:1.